Side-chain cleavage of cholesterol esters by human cytochrome P-450(scc).

In order to define the substrate binding site of human cytochrome P-450(scc) in the vicinity of the 3beta-hydroxyl group of cholesterol, we have tested the ability of the cytochrome to cleave the side chain of a range of cholesterol esters and cholesterol methyl ether. Using a Tween-20 detergent reconstituted system we found that cholesterol sulphate could undergo side-chain cleavage with the same turnover number (kcat) as that for cholesterol, but with a higher Km. Cholesterol methyl ether underwent side-chain cleavage to pregnenolone methyl ether with kcat and Km values 30% of those for cholesterol. Cholesterol fatty acid esters with acyl chain lengths of up to four carbons were able to undergo side-chain cleavage with Km values similar to those for cholesterol, but kcat values only 12-23% of those for cholesterol. Turnover numbers decreased as the acyl group length increased beyond four carbons, although some activity was still detected with cholesterol palmitate as substrate. Analysis of bovine cytochrome P-450(scc) revealed that it could also cleave the side chain of acyl and sulphate esters of cholesterol. This study indicates that the substrate binding site of cytochrome P-450(scc) in the vicinity of the 3beta-hydroxyl group is larger than previously believed.

Assessment of the effect of increased dietary fibre intake on bowel function in patients with spinal cord injury.

It is common for constipation to occur following severe spinal cord injury (SCI). Although a bowel management program including a high fibre diet is an integral part of rehabilitation, the effect of a high fibre diet on large bowel function in SCI has not been examined. The aims of this study were to assess the nutrient intake of SCI patients, to determine baseline transit time, stool weight and evacuation time and to assess the effect of addition of bran on large bowel function. Eleven subjects, aged 32 +/- 10.5 years participated in the study. The level of injury ranged from C4 to T12; only one patient had an incomplete injury. Baseline mean energy intake was 7823 +/- 1443 kJ/d, protein intake 93 +/- 21 g/d, carbohydrate intake 209 +/- 39 g/d and mean dietary fibre intake 25 +/- 8 g/d. Mean baseline stool weight was 128 +/- 55 g/d and bowel evacuation time was 13 +/- 7.4 min/d. Three subjects who consumed < 18 g dietary fibre/d had low stool weights of 60-70 g/d and two had very delayed transit times that were too slow to enable quantitation. Mean mouth to anus transit time was 51.3 +/- 31.2 h, mean colonic transit time 28.2 +/- 3.5 h, right colonic transit time 5.9 +/- 4.5 h, left colonic transit time 14.5 +/- 5.2 h and rectosigmoid colonic transit time 7.9 +/- 5.6 h. Following the addition of bran, dietary fibre intake significantly increased from 25 g/d to 31 g/d (P < 0.001). However, the mean colonic transit time increased from 28.2 h to 42.2 h (P < 0.05) and rectosigmoid colon transit time increased from 7.9 to 23.3 h (P < 0.02). Stool weight, mouth to anus, left and right colon transit time and evacuation time did not change significantly. Results of this study suggest that increasing dietary fibre in SCI patients does not have the same effect on bowel function as has been previously demonstrated in individuals with 'normally functioning' bowels. Indeed the effect may be the opposite to that desired. This preliminary study highlights the need for further research to examine the optimal level of dietary fibre intake in SCI patients.

Cytochrome P-450scc activity and substrate supply in human placental trophoblasts.

The degree of saturation of cytochrome P-450scc with cholesterol and the substrate turnover number of the cytochrome in cultured trophoblasts and mitochondria from the human placenta were investigated. Cholesterol sulfate was found to be a suitable substrate for probing the degree of saturation of cytochrome P-450scc with substrate during culture and in isolated mitochondria, since it enabled the maximum velocity of the cholesterol side-chain cleavage reaction to be estimated. In contrast, 25-hydroxycholesterol and low density lipoprotein supported trophoblast progesterone production at lower rates than that measured with saturating cholesterol sulfate. In the absence of exogenous substrate, the highest rate of progesterone synthesis by trophoblasts was observed at the beginning of the culture. With cholesterol sulfate as substrate, the turnover number of cytochrome P-450scc in cultured cells was 2.8 min-1 and was not significantly different to the turnover number of the cytochrome for placental mitochondria, where cholesterol is known to be saturating. Results indicate that cholesterol is limiting for progesterone synthesis in cultured trophoblasts even in the presence of lipoprotein rich medium and 8-bromo-cAMP. The concentration of cytochrome P-450scc in trophoblasts was only 20% of that measured for placental homogenate suggesting an induction of the cytochrome occurs when the trophoblasts fuse in vivo to form syncytiotrophoblasts.

Cytochrome P-450scc activity and substrate supply in human placental trophoblasts.

The degree of saturation of cytochrome P-450scc with cholesterol and the substrate turnover number of the cytochrome in cultured trophoblasts and mitochondria from the human placenta were investigated. Cholesterol sulfate was found to be a suitable substrate for probing the degree of saturation of cytochrome P-450scc with substrate during culture and in isolated mitochondria, since it enabled the maximum velocity of the cholesterol side-chain cleavage reaction to be estimated. In contrast, 25-hydroxycholesterol and low density lipoprotein supported trophoblast progesterone production at lower rates than that measured with saturating cholesterol sulfate. In the absence of exogenous substrate, the highest rate of progesterone synthesis by trophoblasts was observed at the beginning of the culture. With cholesterol sulfate as substrate, the turnover number of cytochrome P-450scc in cultured cells was 2.8 min-1 and was not significantly different to the turnover number of the cytochrome for placental mitochondria, where cholesterol is known to be saturating. Results indicate that cholesterol is limiting for progesterone synthesis in cultured trophoblasts even in the presence of lipoprotein rich medium and 8-bromo-cAMP. The concentration of cytochrome P-450scc in trophoblasts was only 20% of that measured for placental homogenate suggesting an induction of the cytochrome occurs when the trophoblasts fuse in vivo to form syncytiotrophoblasts.

Side-chain specificities of human and bovine cytochromes P-450scc.

Cytochrome P-450scc catalyses the conversion of cholesterol to pregnenolone by the sequential hydroxylation of the side chain of cholesterol. This occurs at a single active site and produces 22R-hydroxycholesterol and 22R-20 alpha-dihydroxycholesterol as intermediates. To further define the active site of human and bovine cytochromes P-450scc, we have examined the kinetics of the conversion of structural analogues of cholesterol with modified side chains, to pregnenolone. Analysis of the side-chain cleavage of analogues of cholesterol modified at C22 confirmed the high degree of structural specificity for the 22R position by cytochrome P-450scc, the major effect being on the turnover number (kcat) rather than on binding. The analogues of cholesterol that had a polar group at C24, C25 or C26 had much lower Km values and generally lower kcat values than the non-polar analogues which were tested. Km values of the polar analogues were 3-25-times lower than the Km for cholesterol and kcat values were also much lower than the kcat values for cholesterol, particularly for the human enzyme. The data suggest that the tight binding of the analogues with a hydroxyl or ketone group at C24, C25 or C26 places C20 and C22 in a poor orientation relative to the heme group for hydroxylation to occur. Many of the polar analogues which were tested are postulated regulators of cellular cholesterol metabolism. Several of these analogues are good substrates for bovine and human cytochromes P-450scc at low substrate concentration, as determined from their kcat/Km values. This study also indicates that the active site of cytochrome P-450scc is well conserved between bovine and human cytochromes. However, small species differences are evident since lower kcat values relative to the kcat of cholesterol are observed for some polar side-chain analogues of cholesterol with the human enzyme.

Catalytic properties of cytochrome P-450scc purified from the human placenta: comparison to bovine cytochrome P-450scc.

Cytochrome P-450scc was purified from the human placenta by extraction of mitochondria with cholate and Emulgen 911, chromatography on phenyl-Sepharose and DEAE-Sephacel, and ammonium sulphate fractionation. The catalytic properties of the purified human cytochrome P-450scc were analysed in Tween-20 micelles and compared to those of bovine adrenal cytochrome P-450scc analysed in the same system. Both enzymes had the same Km for cholesterol and were stimulated by cardiolipin when the cholesterol concentration was subsaturating. Examination of the rates of pregnenolone synthesis from 20 alpha-hydroxycholesterol, 22R-hydroxycholesterol and 20 alpha, 22R-dihydroxycholesterol by human and bovine cytochromes P-450scc revealed that the first hydroxylation (22R position) was rate-limiting for both in Tween-20 micelles. The rate of the 22R-hydroxylation was further decreased when a 20 alpha-hydroxyl group was already present on the cholesterol side-chain. The second hydroxylation occurred at about the same rate as the third hydroxylation for both enzymes. The rate of side-chain cleavage of 25-hydroxycholesterol by human cytochrome P-450scc in Tween-20 micelles was low, the highest rate being about 1% of the Vmax for cholesterol. Substrate inhibition was seen with high concentrations of 25-hydroxycholesterol. Conversion of 25-hydroxycholesterol to pregnenolone was accompanied by a build-up of products with intact side-chains, which were probably intermediates of the reaction. Side-chain cleavage of 25-hydroxycholesterol by bovine cytochrome P-450scc showed similar characteristics to the human enzyme, except that the highest velocity observed was approx. 25% of the Vmax for cholesterol. Rates of cleavage of 25-hydroxycholesterol by both enzymes were higher in dioleoylphosphatidylcholine vesicles than in Tween-20, but were still well below the Vmax for cholesterol and showed substrate inhibition. This study shows that there is close similarity in catalytic properties between human and bovine cytochromes P-450scc which suggests that the active site of the cytochrome is highly conserved.

Human placental cholesterol side-chain cleavage: enzymatic synthesis of (22R)-20 alpha,22-dihydroxycholesterol.

(22R)-20 alpha,22-Dihydroxycholesterol is the second intermediate in the conversion of cholesterol to pregnenolone by cytochrome P450scc in steroidogenic tissues. We report a rapid method for the enzymatic synthesis of (22R)-20 alpha,22-dihydroxycholesterol from (22R)-22-hydroxycholesterol using mitochondria from the human placenta.

Comparison of IgE expression at the mRNA and protein levels in vitro.

The regulating effects of IL-4 and pokeweed mitogen on IgE synthesis in vitro by human peripheral blood leucocytes has been compared with the corresponding effect of these regulators on the expression of IgE mRNA. The latter was measured by dot blot hybridization with an oligonucleotide coding for a unique six amino acid region of the CH epsilon 2 domain. Specificity of the oligonucleotide probe was established by its inability to hybridize with RNA extracted from HMY-2 (IgG) and XQ-15 (IgM) secreting cell lines whilst producing intense signals with RNA extracted from the IgE secreting cell line U266. Whilst IgE mRNA was detected in RNA extracted from PBL of both atopic and control subjects, spontaneous IgE synthesis was restricted to atopic PBL. IL-4 increased both IgE mRNA and IgE synthesis in all PBL samples but PWM, while significantly increasing IgE mRNA expression either failed to modify IgE synthesis or actively suppressed it. The assay system employed to quantitate IgE synthesis in vitro was shown to be inhibited by both IgE binding factors and IgG anti-IgE autoantibodies which are produced in PBL cultures. IgE mRNA levels might therefore more accurately monitor the regulatory effects of IL-4 and PWM on IgE synthesis than quantitation of the IgE by radioimmunoassay.

In vitro synthesis of IgE by human peripheral blood leucocytes: VI. Seasonal variations in synthesis of rye pollen (Lolium perenne) specific IgE.

Fractionation by Percoll density centrifugation of peripheral blood leucocyte cells, from atopic subjects with seasonal hay fever, unmasked IgE-B cell populations whose individual capacities to synthesize IgE in vitro were obscured in cultures of unfractionated B cells. B cell cultures from all six subjects in the study released rye pollen-specific IgE during the 6 days of culture, but actual synthesis was significant only in October, the pollen season. Synthesis in October occurred most frequently in cultures of mature, low density B cells, which generally responded to the addition of autologous T cells with enhanced synthesis (T-help). T-help was also found for high density B cells in the mid-winter (July) cultures. Total IgE synthesis in vitro demonstrated a less seasonal relationship, although it tended to be maximal for low density B cell cultures in October and for high density B cells in May. All B cell cultures contained preformed total and rye-specific IgE antibody which persisted throughout the pre- and post-pollen seasons, particularly in the low density B cell fractions, even in the absence of de novo synthesis. Moreover, the intracellular levels of rye pollen-specific IgE antibody were often higher in the winter than in the peak of the pollen season. The relevance of this preformed IgE remains to be established.

Ultrastructural localization of IgE in peripheral blood monocytes from atopics.

Monocyte-enriched preparations derived from peripheral blood leukocytes of atopics were probed via a cocktail comprising peroxidase-conjugated (Fab1)2 fragments of two monoclonal antibodies against human IgE. Reaction product indicative of intracellular IgE was identified by electron microscopy in both large and small vacuoles, and at high magnification exhibited a characteristic granular deposition pattern consistent with highly concentrated (perhaps insolublized) material. IgE-containing vacuoles were observed with comparable frequency to those containing IgG, despite the greater than 10,000-fold relative excess of the latter in serum suggesting highly selective uptake of IgE by the monocytes. These results are similar to those reported recently for IgA in human milk macrophages.

In vitro synthesis of IgE by human peripheral blood leucocytes: V. Functional heterogeneity within the IgE-B-cell pool.

Fractionation of human peripheral blood leucocytes (PBL) B cells by differential sedimentation on a discontinuous Percoll gradient separates B cell subpopulations which vary markedly in rates of spontaneous IgE synthesis, often revealing the presence of active IgE secreting cells which are totally suppressed within unfractionated PBL B cell preparations. The production in vitro of IgE by separated B cell populations from the same individual may respond disparately to an identical population of autologous T cells and to pokeweed mitogen. Kinetic studies revealed major differences in both the rates of release of cell-associated IgE between these B cell populations, and their rates of de novo IgE synthesis. From a methodological viewpoint, the use of this B cell fractionation technique is demonstrated to improve greatly the efficiency of detection of T cell-responsive IgE producing B cells in peripheral blood from atopics, and from a mechanistic standpoint raises the possibility that B cell heterogeneity may modulate the functional expression of IgE-regulatory T cells signals.

Functional heterogeneity within the human peripheral blood B cell pool engaged in IgE synthesis.

Fractionation of human peripheral blood leukocytes (PBL) B cells by differential sedimentation on percoll gradients separates B cell subpopulations which vary markedly in rates of spontaneous IgE synthesis, in their response to identical populations of autologous T cells and in their response to soluble T cell factors. This B cell fractionation technique often reveals the presence of active IgE-secreting cells which are totally suppressed within unfractionated PBL B cell preparations and greatly improves the efficiency of detection of T cell-responsive, IgE-producing B cells in PBL of atopics.

Seasonal variations in in vitro synthesis of rye pollen-specific IgE by human peripheral mononuclear cells. Functional heterogeneity within the IgE-B pool.

Peripheral blood mononuclear B cells from 6 rye pollen-allergic patients, with no consistent perennial symptoms, were isolated before (July), during (October) and after (February and May) the pollen season. The T-depleted cells were fractionated on a discontinuous percoll density gradient and the B cell fractions, together with unfractionated B cells, incubated in vitro for quantitation of spontaneous synthesis of rye pollen-specific IgE. Markedly higher levels of IgE were synthesised by the fractions, as opposed to unfractionated B cells. The low-density fraction (B5) contributed most towards synthesis in the pollen season and the denser B6 cells in the pre- and post-pollen season. All low-density B cell fractions (B1-3-B5) and some high-density fractions contained large but variable amounts of preformed specific IgE which was retained, even in the absence of de novo synthesis in vitro, during the post- and pre-pollen season. Since only a fraction of this preformed IgE escaped into culture supernatants the contribution of preformed IgE to in vitro IgE synthesis in general may require reappraisal.

In vitro synthesis of IgE by human peripheral blood leucocytes. IV. Longitudinal study of regulatory T-cell activity in atopics.

The assessment of IgE production in cultures of T- and B-cells from peripheral blood is proving a useful tool to probe IgE immunoregulation in human atopics. The present study contrasts secretion and synthesis as indices of IgE production, and demonstrates that these measures yield comparable data upon the magnitude and direction of regulatory T-cell effects (help vs. suppression) in severe atopics. The majority of peripheral blood B-cell samples from the atopics in this study exhibited spontaneous IgE synthesis and secretion, and in vitro T-cell help and suppression were observed with equal frequency within the sample population. Repeated testing of individual atopics indicated that the direction of T-cell effects remained stable in some (but not all) atopics over periods as long as 3 years.

Enumeration of human immunoglobulin-secreting cells by the ELISA-plaque method: IgE and IgG isotypes.

A new system is described for the enumeration of human immunoglobulin-secreting cells (ISC), based upon the ELISA methodology. In principle, putative ISC are incubated over a solid phase containing bound anti-Ig of the isotype being tested. Secreted Ig is immobilized at or near the point of release from the ISC, and the resulting Ig fingerprint of the ISC is then visualized by the sequential application of an anti-Ig-alkaline phosphatase conjugate, followed by a substrate-agarose overlay. The system is capable of detecting IgE-secreting cells, and pokeweed mitogen-stimulated IgG-secreting cells with sensitivity at least equivalent to the protein A hemolytic plaque assay.

In vitro synthesis of IgE by human peripheral blood leucocytes. III. Release of pre-formed antibody.

The appearance of IgE in supernatant fluids from cultures of human peripheral blood leucocytes (PBL) is employed in many laboratories as an index of antibody 'synthesis'. This study demonstrates that, unlike IgG, the majority of cell associated IgE is sequestered by PBL in a tightly bound form, which resists extraction via the freeze-thawing (F/T) techniques in current use. A method is presented for the quantitative extraction of cell associated IgE, involving brief acid treatment of whole cells, and is shown to yield up to 100% more IgE than the F/T procedure. The use of this extraction process on PBL before and after culture permits discrimination between release of pre-formed IgE and de novo synthesis, both of which are demonstrated to occur to widely differing degrees in PBL cultures from different donors.